Covalent complexes are intermediates in the catalytic cycle of topoisomerases that form when a tyrosine residue from the active site of the enzyme becomes linked to the backbone of DNA. Under normal circumstances the covalent complex is transient, but a wide variety of circumstances lead to its stabilization, and the resulting long-lived disruption in the continuity of the DNA backbone can have dire consequences for genome stability. Since topoisomerases are abundant enzymes, covalent complexes must be common occurrences and, given their potential for stabilization, their repair is likely to be an important part of DNA metabolism. However, such repair presents special problems since the strand break is encumbered with a covalently bound polypeptide which must be removed to restore the continuity of the chromosome. Our laboratory has been interested in the mechanisms by which eukaryotic cells, exemplified by the budding yeast S. cerevisiae, deal with covalent complexes of topoisomerase I (Top1). A defining characteristic of eukaryotic Top1 is the linkage of its active site tyrosine to the 3'-end of DNA. We previously described an enzyme, tyrosyl-DNA phosphodiesterase (Tdp1), that can specifically hydrolyze this linkage. We further showed that genetic inactivation of this enzyme resulted in sensitization of yeast to Top1 damage. Although this served to prove that Tdp1 was involved in repair of topoisomerase damage, the degree of sensitization was modest in comparison with cells defective in all double-strand break repair (DSBR), implying that, in addition to Tdp1, yeast has other ways to remove covalently bound Top1. In the past year, we have evaluated several candidate genes for enzymes that might act in parallel to Tdp1 so as to generate free ends of DNA. In contrast to negative results with genes for the AP endonucleases APN1 and APN2, assays of growth in the presence of the Top1 poison camptothecin (CPT) indicate that the structure-specific nucleases dependent on RAD1 and MUS81 can each contribute independently of TDP1 to repair. The known activity of these enzymes suggest that they act by cutting off a segment of DNA along with the topoisomerase. We conclude that multiple pathways of repair of topoisomerase damage function in parallel with the Tdp1-dependent pathway. It must be emphasized that the list of genetic pathways is still incomplete. Compared to the CPT sensitivity achieved by inactivation of the global repair gene MRE11 (which we show is even more important for repair of topoisomerase damage than the RAD52 gene), the sensitivity of a mus81 rad1 tdp1 triple mutant strain is only partial. There must be additional pathways of repair that can undo topoisomerase damage, albeit slowly, and thereby restore clonogenic capacity once the generation of lesions is terminated. This view provides a potential explanation for the recent finding by others that, in humans, an inherited mutation in TDP1 is associated not with defects in rapidly dividing cells but with gradual loss of post-mitotic neurons. We postulate that, as in yeast, dividing human cells have multiple pathways for repair of topoisomerase damage but many of these are not retained upon terminal differentiation. This leaves Tdp1 as the primary defense against accidental abortion of the topoisomerase enzyme cycle, and its inherited loss leaves postmitotic neurons at risk. Two additional issues concerning Tdp are being addressed in collaborative efforts. One concerns the possibility that the enzyme also plays a role in repair of a lesion significantly different from a 3'-terminal tyrosine. Specifically, studies done principally with human enzyme show that Tdp1 can hydrolyze 3'-terminal phosphoglycolate, the principal lesion induced by the radiomimetic compound bleomycin. In order to examine the biological significance of this activity, we have extended this study to the yeast enzyme. Our biochemical analysis indicated that terminal phosphoglycolate is hydrolyzed some fifty-fold slower than terminal tyrosine. Moreover, genetic inactivation of TDP1 was by itself insufficient to sensitize yeast to the lethal effects of bleomycin. However, such inactivation increased the sensitivity of a strain defective in AP endonucleases. It will require further work to see if this effect of a tdp1 mutation is a direct consequence of diminished phosphoglycolate repair or an indirect consequence of diminished repair of Top1 trapped near bleomycin-induced lesions. The second collaborative effort asks whether Tdp1 is normally associated with other components of repair. We modified the chromosomal locus of yeast TDP1 so as to add a molecular tag to the carboxyl terminus of the protein. At the instigation of our collaborators in LNT/NIMH, we used the recently described Tandem Affinity Purification (TAP) construct, an elegant method that should increase the likelihood of isolating native complexes involving a protein bearing this tag. Extracts of the modified yeast show that the tag does not inactivate the enzyme; pilot studies to look for complexes are under way.